With the advent of recombinant DNA methodology and especially in view of its evident commercial applicability, the ability to synthesize oligodeoxyribonucleotides of defined sequences has become increasingly important.
As now is very well recognized, RNA and DNA are polynucleotides referred to as nucleic acids. The polynucleotides, in turn, are composed of monomers (nucleotides). A nucleotide is a phosphate ester of the N-glycoside of a nitrogenous base and consists of a purine or a pyrimidine base, a pentose (D-ribose in RNA or 2'-deoxy-D-ribose in DNA), and a phosphate group.
Four nitrogenous bases are present in both DNA and RNA. The four present in DNA are: ##STR1## The nitrogenous bases in RNA differ from those in DNA only in that uracil (U) replaces thymine. ##STR2##
The combination of a nitrogenous base at the point of the asterisk (*) in the foregoing formulas with a ribose at its 1'-position is called a ribonucleoside (D-ribose) or a deoxyribonucleoside (2'-deoxy-D-ribose). The corresponding ribonucleotide or deoxyribonucleotide is produced by addition of a phosphate group at the 3'-position of the ribose.
The thus-defined suitably-blocked ribonucleotide or deoxyribonucleotide represents the basic building block in the synthesis of RNA or DNA, respectively. A standard and highly attractive method for synthesizing RNA or DNA is known in the literature as the "triester method". Using the synthesis of a polydeoxyribonucleotide as an example, the procedure involves coupling a mononucleotide or oligonucleotide having a 3'-phosphate diester with a mononucleoside, a blocked 3'-hydroxy oligonucleotide, a mononucleotide, or an oligonucleotide having an available 5'-hydroxyl group. This method can be represented schematically as follows: ##STR3## In the foregoing, B is a nitrogenous base selected from adenine, cytosine, guanine, and thymine, each of the first three having their reactive moieties blocked by suitable protecting groups; R is a blocking group for the 5'-hydroxyl; R.sup.3 is a blocking group for the 3'-hydroxyl cleavable under alkaline conditions or a group of the formula ##STR4## and R.sup.1 and R.sup.2 are selevtively removable groups which block the reactive phosphate moiety.
Additional discussion of the triester method can be found in various publications including, for example, Narang, S. A., Hsiung, H. M., and Brousseau, R., "Improved Phosphotriester Method for the Synthesis of Gene Fragments", Methods in Enzymology, Vol. 68, Academic Press, New York, N.Y., (1979), pp. 90-98; and Narang, S. A., Brosseau, R., Hsiung, H. M., and Michniewicz, J. J., "Chemical Synthesis of Deoxyoligonucleotides by the Modified Triester Method", Methods in Enzymology, Vol. 65, Academic Press, New York, N.Y., (1980), pp. 610-620.
The triester method described above, of course, has been applied in coupling two oligonucleotides, an oligonucleotide and a mononucleotide, or, as specifically illustrated above, two mononucleotides. Whatever the entities, the reaction involves the coupling of an available 5'-hydroxyl with a 3'-phosphate diester group. Moreover, the reactant containing the available 5'-hydroxyl can have a blocked terminal phosphate or such can be lacking (i.e., R.sup.3 is a 3'-hydroxyl blocking group). The two reactants are coupled in the presence of a solvent, typically pyridine, and in the presence of a coupling agent, for example, 2,4,6-trimethylbenzenesulfonyl tetrazolide.
The product from the coupling reaction generally is recoverable from non-coupled nucleotide starting materials using standard and recognized conditions of chromatographic separation. However, in order to render the reaction mixture ready for chromatography, it has been necessary to eliminate amounts of residual coupling agent present in the reaction mixture.
The method heretofore used to render the reaction mixture ready for chromatographic purification involves a complex and tedious sequence initiated by aqueous decomposition of the coupling reagent. In addition to being complex and tedious, the recognized method, due to the conditions employed, generally results in measurable product degradation. In general, the work-up heretofore employed involves (1) addition of water to decompose the coupling agent, (2) concentration of the reaction mixture, (3) addition of chloroform, (4) washing with saturated sodium bicarbonate, (5) drying of organic phase, and (6) evaporation of organics. The residue then is ready for chromatographic purification.
A method for readily and rapidly placing product from a nucleotide coupling reaction into condition for chromatographic purification has now been discovered. It is to such a process that this invention is directed.